Expression, Purification, and Characterization of the Mast Cell Proteases Chymase and Cathepsin G.

<p>Human mast cells have been associated with wound healing, allergies, inflammation, and defense against pathogens and have been detected in tissues close to blood vessels especially in the areas between the inside of the body and the external environment, such as the skin, lungs, digestive tract, mouth, and nose. Previous studies have shown that mast cells contain large granules filled with histamine, heparin, cytokines, eicosanoids, and the serine proteases, tryptase, Chymase, and cathepsin G (CatG). These proteases are stored and released from mast-cell granules upon activation by antigen binding to IgE immunoglobulins on the cell surface or by direct injury. In this study, chymase and CatG were expressed as active enzymes in the yeast <em>Pichia pastoris</em> by homologous recombination of the cDNA coding for the mature active proteases into the <em>Pichia</em> genome. Methanol induction resulted in the secretion of active enzyme into the <em>Pichia</em> growth media and increasing levels of enzyme were detected in the media for 5 days. Cells that secreted the highest levels of activity were selected by kinetic assay. Active chymase was purified from the culture media with a 22% yield of activity by a simple two-step procedure that involved hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media. Active CatG was partially purified from culture media using an ultrafiltration. Mass Spectroscopy (Maldi-Tof) data confirmed that the major protein band was CatG, resulting in the first active human CatG to be produced recombinantly. Additionally, the partially purified enzyme was active against both chymotrypsin and trypsin substrates, and its reaction with inhibitors was consistent with CatG. Although the protein yields were low, these results confirm that CatG was recombinantly expressed.</p>

Name thesis:degree_name

PhD (Doctor of Philosophy)

Level thesis:degree_level

Dissertation - unrestricted

Discipline thesis:degree_discipline

Biomedical Sciences

Year dc:date.issued

2008

Author dc:creator

• Lockhart, Brent E

• chymase
• protease
• recombinant
• Cathepsin G
• Chemicals and Drugs
• Enzymes and Coenzymes
• Medicine and Health Sciences

Statement dc:rights

• Copyright by the authors.

Repository record dc:identifier

https://dc.etsu.edu/etd/1922

OAI identifier oai:identifier

oai:dc.etsu.edu:etd-3274